Antiinflammatory, antinociceptive and antioxidant activities of by NgZcbk7f


									Received by Qing                      Received on 2012-5-24
ID No. B386                           Revised on 2012-5-28
Pages 19
        Antiinflammatory, antinociceptive and antioxidant activities of
                                  Phyllanthus acidus L. extracts

                  Raja Chakraborty*1, Biplab De2, Nayakanti Devanna3, Saikat Sen 4

             Department of Pharmaceutical Chemistry, Creative Educational Society’s College of
                           Pharmacy, Kurnool 518 218, Andhra Pradesh, India.
        Department of Pharmaceutical Science, Assam University, Silchar 788 011, Assam, India.
             Oil Technological Research Institute, JNTU Anantapur, Anantapur 515 001, Andhra
                                             Pradesh, India.
    Department of Pharmacology, Creative Educational Society’s College of Pharmacy, Kurnool
                                     518 218, Andhra Pradesh, India.

* Correspondence address:
               Raja Chakraborty
               Assistant Professor, Department of Pharmaceutical Chemistry,
               Creative Educational Society’s College of Pharmacy,
               NH 7, Chinnatekur, Kurnool, Andhra Pradesh – 518218, India.
               Tel.: +919494222701
               E-mail address:
Objective: To evaluate analgesic, anti-inflammatory and in vitro antioxidant potential and
determine total phenolic, total flavonoid content of leaves extracts of Phyllanthus acidus, a
folk medicinal plant of India.
Methods: Anti-inflammatory activity was evaluated using carrageenan induced paw oedema,
cotton pellet induced granuloma, membrane stabilizing activity method. Analgesic activity of
the extracts was estimated against acetic acid induced writhing, tail immersion method,
formalin test. Free radical scavenging and antioxidant potential of the extracts of Phyllanthus
acidus leaves was performed using several in vitro and ex vivo assay models. Total phenolic
and total flavonoid contents of the extracts were determined using standard chemical
Results: The extracts exhibited significant anti-inflammatory and analgesic activities at dose
dependent manner. Methanol extract at a dose of 500 mg/kg showed superior activity which
was comparable with the standard drugs. Ethyl acetate extract showed moderate activity
while petroleum ether extract showed least activity. Total phenolic and total flavonoid
content in methanol extract were 73.08±0.682 mg GAE/g and 61.28±0.062 mg QE/g
respectively. The extracts possess significant antioxidant activity, methanol extract showed
highest IC50 value. The contents of flavonoids and phenolic compounds could be correlated
with the antioxidant, analgesic and anti-inflammatory activities observed for Phyllanthus
acidus leaves.
Conclusion: Our findings suggest that Phyllanthus acidus contains potential antioxidant,
analgesic and anti-inflammatory compounds which could be tested as drug candidates against
oxidative stress, pain and inflammation related pathological diseases.

Keywords: Phyllanthus acidus, leaves, analgesic, anti-inflammatory, antioxidant, phenolic
content, flavonoid content, methanol extract.
1. Introduction
         Phyllanthus acidus Skeels belonging to the family Phyllanthaceae is distributed commonly
through out India, and often used by ethnic peoples of north-east India in folk medicine. P acidus
have been used traditionally in the treatment of several pain, inflammatory and oxidative stress related
disorders such as rheumatism, bronchitis, asthma, respiratory disorder, hepatic disease, diabetes and
gonorrhoea. The plant is also important to improve eyesight, memory and to cure cough, psoriasis,
skin disorders, sudorific[1,2,3]. Fruits of the plant used as astringent, root and seed are useful as
cathartic, leaf and root use as antidote to viper venom[4]. The leaf of the plant found effective in
hypertension[5]. Leaf, bark and roots are used to treat fever traditionally[6] The latex of the plant
credited with purgative and emetic activity[7].
         Methanolic extract of fruit and leaves was reported to possess antimicrobial activity[8]. In vitro
screening of petroleum ether extract of fruits showed cytotoxic, antibacterial and antioxidant
activity[9]. Different parts of P. acidus have been reported for several biological activities, fruits and
leaves of the plant showed promising hepatoprotective activity[10]. Phyllanthosols A and B were
isolated from roots, which were proposed as promising antitumor activity[11]. Adenosine, kaempferol,
and hypogallic acid were found in leaves which showed airway chloride secretion, a potential
treatment in cystic fibrosis[12].
         Phyllanthus genius is reach in different secondary metabolite like alkaloids, tannins,
flavonoids, lignans, phenolics and terpenes[13]. Several species of phyllanthus have shown
antinociceptive activity in mice[14].
         In this study, P. acidus leaves extracts were assessed for analgesic, antiinflamatory and
antioxidant activity to find credence of the traditional usage of P. acidus in antiinflamatory and
oxidative stress related disorder. In addition, we measured the content of total phenolic compounds
and flavonoids, in the extracts in order to correlate them with the assayed activities.

2. Materials and methods
2.1. Plant materials
      The leaves of Phyllanthus acidus was collected in February 2011 from Tripura, India.
The plant identified as Phyllanthus acidus L. Skeels was confirmed by Dr. B. K. Datta,
Department of Botany, Tripura University, Tripura, India. A voucher specimen was deposited
in Plant Taxonomy & Biodiversity Laboratory, Tripura University for further reference.
2.2. Drugs and chemicals
       Indomethacin capsule, diclofenac sodium, aspirin was obtained from Ranbaxy
Laboratories Limited (India). Acetic acid, 2,2-diphenyl-picrylhydrazyl (DPPH), phenazine
methosulfate (PMS), and nitro blue tetrazolium (NBT), Folin–Ciocalteau reagent, ferrozine,
butylated hydroxyanisole (BHA), ascorbic acid were purchased from              Sigma Aldrich
(Bangalore, India). Trichloro acetic acid (TCA), thio barbituric acid (TBA), 2-deoxy-ribose,
and quercetin, α-tocopherol, and gallic acid were procured from SD Fine Ltd. Mumbai.
Linoleic acid, All other chemicals used in the study were obtained commercially and were of
analytical grade.

2.3. Preparation of extracts
       Air dried fine leaf powder extracted using methanol, ethyl acetate, petroleum ether
separately for 14 h and concentrated yield was 17.7%, 16.2%, 10.6% respectively, which
were then stored at 4ºC until the time of use.

2.4. Animals
       Wistar rats and albino mice used to different analgesic, anti-inflammatory and lipid
peroxidation inhibition assay. Animals were housed under standard environmental conditions
(24±1°C) with 12 h light – 12 h dark cycles. The study was approved by the Institutional
Animal Ethical Committee (Reg. no: 1305/ac/09/CPCSEA).

2.5. Animal groups
       Healthy adult rats or mice were divided into 8 groups each consists 6 animals for each
analgesic and anti-inflammatory test in following manner

Group I (Control)    : Sodium CMC solution (0.5%).
Group II (Standard) : Indomethacin (10 mg/kg, p.o.) or diclofenac sodium (10 mg/kg, p.o.)
Group III & IV       : Methanol extract of P. acidus (250 mg/kg, 500 mg/kg p.o.).
Group V & VI         : Ethyl acetate extract of P. acidus (250 mg/kg, 500mg/kg p.o.).
Group VII & VIII     : Petroleum ether extract of P. acidus (250 mg/kg, 500 mg/kg p.o.).

2.6. Nociceptive tests
2.6.1. Writhing reflex induced by acetic acid in mice
       The antinociceptive activities of P. acidus extracts were determined in mice using the
writhing test[15]. The each mouse injected with acetic acid (0.6%, v/v, 10 ml/kg), and the
intensity of nociceptive behaviour was quantified by counting the total number of writhes
over a period of 25 min. The extracts, indomethacin, and vehicle were administered orally 1 h
prior to acetic acid injection. The percentage analgesic activity was calculated as follows:
                       Percentage analgesic activity = [(Nc − Nt)/ Nc] × 100%
       Where Nc is the average number of stretches of the control group, and Nt is the
average number of stretches of the test drug group.

2.6.2. Tail immersion test
       Tail immersion test was performed by immersing extreme 3 cm of the albino mouse
tail in a hot water at a temperature of 55±0.5ºC[16]. Within few second each mouse was
reacted by withdrawing the tail, and the reaction time was recorded with a stopwatch. The
drugs were given orally to the respective groups. The experiment was repeated at 0, 0.5, 1, 2,
3, 5 h following the administration of extracts and standard drug.

2.6.3. Formalin-induced licking response in mice
       One hour after oral administration of vehicle, test samples and diclofenac sodium (10
mg/kg), 25 μL of 1% formalin in saline was injected subcutaneously into the subplantar
region of right hind paw. The mice were immediately placed in a clear jar after and the time
spent on licking the injected paw was recorded. The first period (early phase) was recorded at
0-5 min and the second period (late phase) was recorded at 10-35 min[17].

2.7. Anti-inflammatory activity
2.7.1. Carrageenan-induced paw oedema in rats
       Carrageenan induced paw inflammation in rats was produced by injecting carrageenan
(1% solution, 0.1 mL) subcutaneously into the plantar surface of left hind paw of each rat
after administration of respective drug treatment to each group. The volume of the rat paws
was measured with a plethismometer before and 1, 2, 3, 4, and 5 h after [18].

2.7.2. Granuloma formation induced by cotton pellet in rats
       A sterilized cotton pellet of 30 mg was put subcutaneously into the interscapular area
of anaesthetized rats. Male rats were anesthetized using 25 mg/kg of pentobarbitone sodium.
Under sterilized conditions, cotton pellets of 30 mg were implanted subcutaneously in the
interscapular area. The extract solution, indomethacin (5 mg/kg, p.o.), and vehicle water were
administered once daily for 5 consecutive days. On the 5th day, animals were killed via
cervical dislocation after 1h of drug treatment. The cotton pellets with the granuloma tissue
around them were dissected out carefully. The weights of these pellets (wet and dry) were
measured and the anti-proliferative effects of extracts and indomethacin were determined by
comparing with control group[19].

2.7.3. Membrane stabilizing activity
       The blood was collected from rats under mild anaesthesia and mixed with heparin to
prevent clotting. The blood centrifuged was washed three times using 0.9% saline. The 10
mM sodium phosphate buffer (pH 7.4) was used to reconstitute 40% v/v suspension of
erythrocyte and used for this test. The heat induced haemolysis and hypotonic solution
induced haemolysis assay was performed to access membrane stabilizing activity as per the
standard procedures[20].

2.8. Estimation of total phenolic component and total flavonoid content
       The total phenolic content was determined using Foline-Ciocalteu reagent method and
expressed as mg gallic acid equivalents (GAE)/g dry extract[21]. The total flavonoid content of
extracts was determined following a colorimetric method and values were expressed as mg
quercetin equivalent (QE)/ g dry extract[22].

2.9. Antioxidant activity
2.9.1. DPPH radical scavenging assay
       Free radical scavenging capacity of P. acidus leaf extracts was determined in terms of
hydrogen donating or radical scavenging ability of extracts, using the stable DPPH radical [23].
The scavenging activity of extracts was calculated based on the percentage of DPPH radical
scavenged using the equation (1)
       % inhibition = [(Acontrol – Asample)/Acontrol] × 100
       Where, Asample is the absorbance of a sample solution and Acontrol is the absorbance of
the control solution (containing all reagents except the test sample).

2.9.2. Hydrogen peroxide scavenging activity
       Scavenging activity of hydrogen peroxide by the plant extract was determined by the
method of Bozin et al.[24]. Gallic acid was used as standard, percentage inhibition of extracts
and standard compounds were calculated using the equation 1.

2.9.3. Superoxide anion-scavenging activity
       The superoxide anion radical scavenging activity of extracts and BHA was performed
according to a NTB reduction method[25]. In this assay, non enzymatic phenazine
methosulfate - nicotinamide adenine dinucleotide (PMS/NADH) system generates superoxide
radicals, which reduce nitro blue tetrazolium (NBT) to a purple formazan. The scavenging
activity was calculated by the equation (1).

2.9.4. Hydroxyl radical-scavenging activity
       The effect of extracts on hydroxyl radicals was assayed by using the colorimetric
deoxyribose method[26]. In this method, 2-deoxyribose is degraded on exposure to hydroxyl
radicals generated from the Fe3+/ascorbic acid/ EDTA/ H2O2 system, and degraded to
malondialdehyde (MDA). Percentage scavenging activity of extracts and standard quercetin
was measured to find IC50 value.

2.9.5. Nitric oxide radical scavenging assay
       The method of Yen et al.             was adopted to determine the nitric oxide radical
scavenging activity of extracts of P. acidus. Briefly, 4.0 mL extract solution at different
concentrations mixed with 1.0 mL of 25 mM sodium nitroprusside solution and incubated at
37ºC for 2 h. Two millilitre of incubated solution was mixed with 1.2 mL Griess reagent and
absorbance was measured at 570 nm. The experiment was performed (in triplicate) and
percentage scavenging activity was calculated using the Equation (1).

2.9.6. Ferric thiocyanate method
       The antioxidant activity of extracts and standard α-tocopherol was determined
according to the ferric thiocyanate method[28]. All data on total antioxidant activity are the
average of triplicate experiments. The inhibition percentage of lipid peroxidation in linoleic
acid emulsion was determined by equation (1).

2.9.7. Reducing power ability
       Reducing power was measured as per the method performed by Pal et al.                   . The
reducing capacity of ascorbic acid (25-400 µg/mL) was also determined. The test was run in
triplicate and averaged. Increased absorbance indicated increased reducing power of extracts.

2.9.8. Ferrous ions (Fe2+) chelating activity
       The extracts and chelating standard α-tocopherol were assessed for their ability to
compete with ferrozine for iron (II) ions in free solution. The chelating ability of ferrous ions
by the P. acidus leaves extracts was estimated by the method of Gulcin[30]. The inhibition
percentage of ferrozine–Fe2+ complex formation was estimated by using the equation (1).

2.9.9. Lipid peroxidation assay
       Inhibition of lipid peroxidation was assayed by using brain tissue of mice according to
the method described by Tai et al.[31]. The formation of MDA, a cytotoxic product of lipid
peroxidation reaction is widely used to remark the oxidation, which was measured at 532 nm

2.10. Statistical analysis
       Data are given as mean±SEM (for invivo experiments n =6, for invitro experiment n
=3), statistical comparisons were made using one way ANOVA followed by Turkey Multiple
range test. P < 0.05 was considered as significant. For in vitro antioxidant experiments the
concentration of the extract needed to produce a 50% effect (IC50) was calculated graphically.

3. Results
3.1. Analgesic activity of P. acidus leaves
       Methanol, ethyl acetate and petroleum ether extract at a dose of 500 mg/kg
demonstrated a significant analgesic effect against acetic acid induced writhing, inhibiting
pain by 85.12%, 59.99%, and 26.81% as compared to the control respectively (Table 1).
Indomethacin at 5 mg/kg had 83.84% (p<0.001) inhibition of writhing response. Lower dose
of petroleum ether extract did not produced significant inhibition of pain response.
       After a latency period of 0.5 h following oral administration of the extracts at a dose
of 250 and 500 mg/kg, reduction of painful sensation was observed against tail immersion
test and the effect was dose dependent. The significant inhibition was of painful reaction was
observed 1 h after drug administration. The analgesic effects of the extracts became
pronounced between 1 and 3 h post-dosing and but activity decreased after 5 h. Higher dose
of methanol extract had similar activity to that of morphine between 1-3 h. Standard drug
morphine produced significant activity up to 5th h after drug administration (Table 1). Results
represent significant activity of extracts though the duration of analgesic activity was less
than the standard.
       In first phase of formalin induced pain model, methanol, ethyl acetate and petroleum
ether at 500 mg/kg produced 69.28, 60.33, 47.28% inhibition of pain response, while at
second phase the inhibition was 67.10, 62.89, 53.49% respectively. We found that P. acidus
produced antinociceptive activity both in the early and late phases of formalin test (Figure 1).
Results showed that methanol extract at 500 mg/kg produced better effect, therefore extract
exert its analgesic effect through both peripheral and central mechanism.

3.2. Anti-inflammatory activity leaves of P. acidus
       In the present study, an attempt has been made to evaluate the anti-inflammatory
activity of P. acidus leaves by using carrageenan induced paw oedema, cotton pellet
granuloma model. The results are presented in Table 2. All the extracts showed reduction in
carrageenan-induced paw oedema in dose dependent manner. Highest activity was produced
at 5th h. Methanol extract (500 mg/kg) produced highest activity. After 5th h percentage
inhibition by methanol extract (500 mg/kg) was 90.91 %, while indomethacin (5 mg/kg)
produced 96.46 % inhibition. Extract also produced significant anti-inflammatory activity
against cotton pellet induced inflammation. Lower dose of ethyl acetate and petroleum ether
extract did not produce significant inhibition of granuloma formation. Inhibition rate for
methanol extract (500 mg/kg) and indomethacin (5 mg/kg) were 34.22% and 35.21% for wet
weight and 22.04% and 25.86% for dry weight respectively.
       To confirm the membrane stabilizing action of P. acidus leaves extract heat and
hypotonic solution induced haemolysis of erythrocyte membrane assay were performed.
Extracts were able to protect erythrocytes against haemolysis in a dose dependent manner and
the protective effect of methanol extract was better then the NSAID, aspirin (Figure 2). The
percentage inhibition of methanol extract at 100 and 200 µg/ml concentration were 78.13%,
86.09%, 53.58%, 58.49% against heat and hypotonic solution induced haemolysis

3.3. Polyphenolic content of extracts of P. acidus leaves
       The quantity of total phenolic content present in different extracts of P. acidus leaves
was determined from gallic acid calibration curve (Y = 0.0044x + 0.031, R2 = 0.9995), while
calibration curve of quercetin, with the regression equation Y = 0.0288x + 0.0058, R2 =
0.9991 was used to estimate total flavonoid content. Quantity of total phenolics and total
flavonoids content among different extracts were significantly varies (Table 3). The phenolic
and flavonoid content in methanol extract were 73.08±0.682 GAE mg/g of dry material and
61.28±0.062 QE mg/g of dry material, which was highest compare to other extracts.

3.4. Free radical scavenging and antioxidant activity
       Free radical scavenging and antioxidant activity of the extracts were evaluated by
different invitro and exvivo model.
       P. acidus leaves extracts possess a concentration–response relationship in DPPH
radical scavenging activity. The ethyl acetate extract had highest DPPH scavenging effect
compare to other extracts, with an IC50 value of 28.6±0.72 µg/mL. Standard antioxidant
ascorbic acid showed high scavenging activity (IC50=3.3±0.01 µg/mL). The IC50 value of
methanol and petroleum ether extract was 86.0±1.03 and 117.4±1.33 µg/mL respectively
(Table 4).
       P. acidus leaves extracts are capable to inhibit the formation of blue NBT, which was
the indication of superoxide anion scavenging activity of extracts at concentration of 20-160
µg/mL. The IC50 value of methanol, ethyl acetate and petroleum ether extracts was found
21.7±0.09, 71.8±0.39, 31.7±0.11 µg/mL, and BHA (standard drug) showed IC50 value
23.8±0.89 (Table 4).
       The degradation of deoxyribose by Fe+3-ascorbic acid-EDTA-H2O2 system was
significantly (P<0.01) decreased by all extracts of P. acidus tested in hydroxyl radical
scavenging assay model. IC50 value was higher for methanol extract (17.2±0.13 µg/mL) than
the other two extracts and standard drug (Table 4). At 80 µg/mL concentration methanol
extract produced 86.34% scavenging activity for hydroxyl radical, while at same
concentration quercetin produced 90.13% scavenging effect.
       P. acidus leaves extracts significantly decreased the generation of NO radical in the
assay system. Methanol extract exhibited superior NO scavenging activity with an IC50 of
13.0±0.06 µg/mL than the ethyl acetate (IC50=49.8±0.19 µg/mL), petroleum ether extract
(IC50=100.0±0.28 µg/mL) and standard drug ascorbic acid (IC50=23.0±0.08 µg/mL) (Table
       The ability of extracts and positive control gallic acid to scavenge hydrogen peroxide
is shown in Table 4. All extracts produced a dose-dependent hydrogen peroxide scavenging
activity (IC50 was 230.0±3.03 µg/mL for methanol extract, 333.8±3.90 µg/mL for ethyl
acetate extract and 297.6±3.32 µg/mL for petroleum ether extract), though IC50 value of
gallic acid was 65.0±1.32 µg/mL. Moderate activity was exhibited by all extract when
compared to gallic acid.
       In reductive ability method, the extracts cause the reduction of the Fe3+/ferricyanide
complex to the ferrous form. Figure 1 shows the dose-response curves for the reducing
powers of the leaf extracts from P. acidus. The sequence for reducing power was ascorbic
acid > ethyl acetate extract > methanol extract > petroleum ether extract. The reducing power
of methanol, ethyl acetate and petroleum ether extracts increased from 0.804±0.003,
0.785±0.005 and 0.588±0.007 respectively at 50 µg/mL to 1.383±0.006, 1.479±0.006 and
1.180±0.007 at 800 µg/mL respectively.
       Result suggests that extracts had high to moderate levels of ferrous ion chelating
activity in concentration dependent manner. The sequence for chelating power was α-
tocopherol > methanol extract > petroleum ether extract > ethyl acetate extract. The IC50
value of iron chelating activity for the methanol, ethyl acetate, petroleum ether extract and α-
tocopherol was 121.7±1.39, 178.3±2.01, 159.7±1.98 and 107.2±1.23 µg/mL (Table 4).
       The total antioxidant activity of the extracts was determined by the FTC method. The
effects of various solvent extracts of P. acidus leaves in preventing the linoleic acid
peroxidation are shown in Figure 2. On 9th day, the formation of peroxides was stopped
because of non-availability of linoleic acid. But at the presence of extracts the oxidation of
linoleic acid was slow, which clearly showed that all extracts exhibited significant (P<0.05)
antioxidant activity. Methanol extract produced better activity than standard, while ethyl
acetate and petroleum ether extract showed moderate antioxidant activity.
       Malondialdehyde (MDA), a cytotoxic product generated during lipid peroxidation and
used to observed the oxidation. Table 4 showed that methanol extract possess better
inhibition of MDA formation in liver tissue homogenates than other extracts and rutin. IC50
value of rutin, methanol, ethyl acetate and petroleum ether extract was 75.9±0.92, 58.9±0.77,
115.8±1.01, and 138.5±1.31 µg/mL respectively. These data suggested that the crude extracts
of this plant leaf significantly (P < 0.005) inhibited the formation of MDA in liver tissue.

4. Discussion

       Tail immersion method was selected to investigate central analgesic activity. The
drugs acting against tail immersion induced pain attributed their actions through mu (µ)
opioid receptors rather than kappa (κ) and delta (δ) receptors[16]. Acetic acid induced writhing
                                                                                [15, 32]
response was selected to find peripheral analgesic effect of extract                       . Acetic acid
responsible for increase in the peritoneal fluids of PGE2 and PGF2α, serotonin, and
histamine[15]. Acetic acid induced writhing test presents a good sensitivity but poor
specificity. Therefore to get specific result and to avoid misinterpretation of the results, the
formalin test was carried out which has two distinctive phases that can possibly indicate
different types of pain. The early phase reflects centrally mediated pain, which was a result of
direct stimulation of nociceptors; the late phase pain is caused by local inflammation with a
release of inflammatory and hyperalgesic mediators. Therefore this model is useful not only
to screen the analgesic substances, but also for elucidating the mechanism of analgesia[32,33].
Results showed that methanol extract produced better activity and the effect of extract may
medicated through both peripheral and central mechanism.
        The carrageenan induced paw oedema is frequently used as an experimental model of
acute inflammation, while cotton pellet granuloma model was used to measure granuloma
formation in the proliferative phase or in chronic inflammation[19,34]. The carrageenan
induced paw oedema shown biphasic response; first phase is mediated by release of
histamine and serotonin while the second or delayed phase is related to neutrophil infiltration
and release of other neutrophil derived mediators, eicosanoid release, and production of free
            [18, 35]
radicals               . The release of kinin like substances (e.g. bradykinin) involved in oedema
produced in between early and late phase, which later induces the biosynthesis of
prostaglandin and other autacoids, which are responsible for formation of the inflammatory
             [18, 36]
exudates                . Extracts specifically methanol extracts found effective in both models and
results were almost similar to that of standard. Therefore chemical constituents present in
extracts might be effective against both acute and chronic inflammation.
        A protective effect on heat and hypotonic solution induced erythrocyte lysis is
considered as a biochemical index of anti-inflammatory activity[18,20]. Since there is a close
resemblance of the RBC membrane system to the lysosomal membrane system, defence
against hypotonicity or heat induced RBC lysis is often seen as an indication of stabilization
of lysosomal membranes, and used as a index of anti-inflammatory activity[18,37]. Further,
plants with membrane stabilizing properties can interfere with the early phase of
inflammatory mediator release, specifically by mediating the release of phospholipase A2 that
stimulates the inflammatory mediator release[18,39]. Therefore, the membrane stabilizing effect
mediated by P. acidus leaves extract may contribute to the anti-inflammatory activity of
       The DPPH radical scavenging assay is based on the decolourization of DPPH by the
antioxidants compound present in extracts[40]. Therefore, DPPH radical scavenging effect of
extract might be attributed to a direct role in trapping free radicals by donating hydrogen
atom. Superoxide anion radical is considered as a one of the strongest reactive oxygen
species among the free radicals, responsible for generation of active reactive species like
hydrogen peroxide and singlet oxygen. These radicals can liberate highly reactive hydroxyl
radical through Fenton-chemistry, thereby initiate lipid peroxidation[21,30]. Hydroxyl radical is
the most reactive radical responsible for enormous biological toxicity and cell damage
through lipid peroxidation           . NO has been associated with a number of physiological
processes in the human and plays an important role in respiratory, immune, neuromuscular
functions. NO act as an atypical neural modulator which is involved in neurotransmitter
release, neuronal excitability and learning and memory. It also participates in diverse
pathological condition including muscle diseases, inflammatory bowel disease, sepsis and
septic shock, primary headaches, HIV-associated dementia, multiple sclerosis and stroke[42].
Strong scavenging of superoxide, hydroxyl and nitric oxide radical will be helpful in
protecting free radical induced diseases.
       Hydrogen peroxide can be formed in vivo by different enzymes like superoxide
dismutase. Inactivation of deferent enzymes usually by oxidation of essential thiol groups
was caused by H2O2. However, H2O2 can be highly toxic in presence of Fe2+ or Cu2+, and can
initiate hydroxyl radical generation through Fenton reaction[25,41,43,44]. Extract significantly
inhibit generation of hydrogen peroxide. The reducing power ability of a compound may
afford key sign about the antioxidant ability of the compound. Reducing power abilty of
compound be linked with the presence of reductones[25,28,45]. Therefore, in this study, the
antioxidant activity of the extracts may be related to its reductive activity.
       The ferrous state of iron is highly reactive and can induce lipid oxidation by breaking
down hydrogen and lipid peroxides to reactive free radicals via the Fenton reaction very
quickly[45,46]. Ferrozine can quantitatively form complexes with ferrous state of ion, but
chelating agents can inhibit this complex formation[25,41,47]. All extracts and standard
antioxidant α-tocopherol interfered with ferrous and ferrozine complex the formation
effectively. FTC method was used to evaluate the ability of extrcts/compound to scavenge
peroxyl radicals during polyunsaturated fatty acids (PUFA) oxidation was measured. A red-
colored complex which has a maximum absorbance at 500 nm was formed during the
reaction between ferric ion and thiocyanate[28,48]. Extracts showed significant total antioxidant
capacity tested against FTC method.
       Lipids such as free and ester forms of polyunsaturated fatty acids and cholesterol are
more vulnerable to the attack of reactive species. Lipid peroxidation can induce disruption
membrane transport proteins and deactivation of membrane-associated enzymes, generates
potentially toxic products, damage to genomic and mtDNA, and ultimately may lead to
unstable cytological conditions such as apoptosis or tumour generation[31,49]. Results showed
that extract have potential to inhibit lipid peroxidation significantly, implying their beneficial
effect against cell membrane lipid oxidation.
       Polyphenolic compounds like phenolic and flavonoid component are important for
their diverse pharmacological action including antioxidant, antimutagenic and in other
diseases caused by oxidative stress. Hydroxyl groups present in the phenolic compounds are
important because of their scavenging ability[39,50]. Result indicates that polyphenolic
component present may responsible for its activity.

5. Conclusion

The present study demonstrated that methanol extract obtained from the leaves of
Phyllanthus acidus presented the potential antioxidant, anti-inflammatory and anti-
nociceptive activities, which are comparable with the reference drugs. This might be
correlated with the presence of phenolic constituents and flavonoids in the extract. Due to the
remarkable analgesic, antiinflammatory and antioxidant activity of the plant, further studies
are in progress in our laboratory for the isolation and identification of the bioactive

Conflict of interest statement
We declare that we have no conflict of interest.

The article is a part of ongoing research work (Ph.D) under JNT University Anantapur,
Andhra Pradesh, India. The authors would like to express thanks to CES College of
Pharmacy for providing laboratory facilities to perform the above work.

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Figure Legends:
Figure 1. Effects of extracts from P acidus leaves and diclofenac sodium (10 mg/kg) on
the formalin-induced licking response in mice. Values are mean±S.E.M. (n=6). *P<0.05,
 P<0.01, ***P<0.001 significantly different from control group (ANOVA followed by
Tukey's test).

Figure 2. Membrane stabilizing activity of P acidus leaves extracts. (A) Heat induced
haemolysis assay, (B) Hypotonic solution induced haemolysis assay. Values are
mean±S.E.M. (n=6). *P<0.05, **P<0.01, ***P<0.001 significantly different from control
group (ANOVA followed by Tukey's test).

Figure 3: Reducing power ability of different leaves extracts from the P acidus at
different concentrations. Results are of triplicate measurements.

Figure 4. Antioxidant activity of P acidus leaves extracts by the FTC method. Results are
of triplicate measurements.

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